Surface plasmon resonance

P. Anton van der Merwe

1. INTRODUCTION 3

2. PRINCIPLES AND APPLICATIONS OF SURFACE PLASMON RESONANCE. 4

2.1. Principles 4

2.2. Applications 5 2.2.1. What SPR is good for 5 2.2.2. What SPR is not good for. 7

3. GENERAL PRINCIPLES OF BIACORE EXPERIMENTS 8

3.1. A typical experiment 8

3.2. Preparation of materials and buffers 9

3.3. Monitoring the Dips 9

4. LIGAND 10

4.1. Direct versus indirect immobilisation 10

4.2. Covalent immobilisation 11 4.2.1. A general approach 11 4.2.2. Choice of chemistry 12 4.2.3. Prepare the protein 12 4.2.4. Pre-concentration 12 4.2.5. Amine coupling 14 4.2.6. Regeneration 15 4.2.7. Adjusting the immobilisation conditions. 16

4.3. Non-covalent immobilisation (ligand capture) 16 4.3.1. Using an existing strategy 17 4.3.2. Developing a new strategy 17

4.4. Activity of immobilised ligand 18

4.5. Control surfaces 19

4.6. Re-using sensor chips 19

5. ANALYTE 20

5.1. Purity, activity and concentration 20

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5.2. Valency 20

5.3. Refractive index effect and control analytes 21

5.4. Low molecular weight analytes 22

6. QUALITITATIVE ANALYSIS; DO THEY INTERACT? 22

6.1. Positive and negative controls 22

6.2. Qualitative comparisons using a multivalent analyte 23

7. QUANTITATIVE MEASUREMENTS 23

7.1. Affinity 24 7.1.1. Concepts 24 7.1.2. Experimental design 24 7.1.3. Data analysis 26 7.1.4. Controls 27 7.1.5. Non-linear Scatchard Plots 27

7.2. Kinetics 30 7.2.1. Concepts 30 7.2.2. Experimental design 31 7.2.3. Data analysis 33 7.2.4. Controls 34

7.3. Stoichiometry 35

7.4. Thermodynamics 35

7.5. Activation energy 37

8. CONCLUSION 38

9. APPENDIX 38

9.1. Physical basis of SPR 38

9.2. Samples and buffers 39 9.2.1. Guidelines for preparing samples and buffers for use on the BIAcore 39 9.2.2. Standard buffers 40

9.3. Additional information 41

10. REFERENCES 41

11. ACKNOWLEDGEMENTS 42

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12. GLOSSARY 42

13. FIGURE 1. ANALYSIS OF DIPS. 44

14. TABLES 45

14.1. Table 1. BIAcore instruments currently available (January 1999) 45

14.2. Table 2. Sensor chips available for use on the BIAcore 46

14.3. Table 3. Covalent coupling chemistry 47

14.4. Table 4. Techniques for ligand capture. 48

14.5. Table 5. Trivial causes of complex binding kinetics 49

14.6. Table 6. Distinguishing some non-trivial causes of complex kinetics 50

1. Introduction

Since the development almost a decade ago (1,2) of the first biosensor based on surface plasmon

resonance (SPR), the use of this technique has increased steadily. Although there are several SPR-

based systems (3-5), by far the most widely used one is the BIAcore (1,2), produced by BIAcore AB,

which has developed into a range of instruments (Table 1). By December 1998 over 1200

publications had reported results obtained using the BIAcore. It is likely that it would be even more

widely used were it not for its high cost and the pitfalls associated with obtaining accurate

quantitative data (5-8). The latter has discouraged many investigators and led to the perception that

the technique may be flawed. This is unjustified because the pitfalls are common to many binding

techniques and, once understood, they are easily avoided (4,8,9). Furthermore, the BIAcore offers

particular advantages for analysing weak macromolecular interactions, allowing measurements that

are not possible using any other technique (4,10). This Chapter aims to provide guidance to users of

SPR, with an emphasis on avoiding pitfalls. No attempt is made to describe the routine operation and

maintenance of the BIAcore, as this is comprehensively described in the BIAcore instrument manual

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(see Section 9.3). Although written for BIAcore users, the general principles will be applicable to

experiments on any SPR instrument.

2. Principles and applications of Surface Plasmon Resonance.

2.1. Principles

The underlying physical principles of SPR are complex (see Section 9.1). Fortunately, an adequate

working knowledge of the technique does not require a detailed theoretical understanding. It suffices

to know that SPR-based instruments use an optical method to measure the refractive index near

(within ~300 nm) a sensor surface. In the BIAcore this surface forms the floor of a small flow cell,

20-60 nL in volume (Table 1), through which an aqueous solution (henceforth called the running

buffer) passes under continuous flow (1-100 µL.min-1). In order to detect an interaction one molecule

(the ligand) is immobilised onto the sensor surface. Its binding partner (the analyte) is injected in

aqueous solution (sample buffer) through the flow cell, also under continuous flow. As the analyte

binds to the ligand the accumulation of protein on the surface results in an increase in the refractive

index. This change in refractive index is measured in real time, and the result plotted as response or

resonance units (RUs) versus time (a sensorgram). Importantly, a response (background response)

will also be generated if there is a difference in the refractive indices of the running and sample

buffers. This background response must be subtracted from the sensorgram to obtain the actual

binding response. The background response is recorded by injecting the analyte through a control or

reference flow cell, which has no ligand or an irrelevant ligand immobilized to the sensor surface.

One RU represents the binding of approximately 1 pg protein/mm2. In practise >50 pg/mm2 of

analyte binding is needed. Because is it very difficult to immobilise a sufficiently high density of

ligand onto a surface to achieve this level of analyte binding, BIAcore have developed sensor

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surfaces with a 100-200 nm thick carboxymethylated dextran matrix attached. By effectively adding

a third dimension to the surface, much higher levels of ligand immobilisation are possible. However,

having very high levels of ligand has two important drawbacks. Firstly, with such a high ligand

density the rate at which the surface binds the analyte may exceed the rate at which the analyte can

be delivered to the surface (the latter is referred to as mass transport). In this situation, mass transport

becomes the rate-limiting step. Consequently, the measured association rate constant (kon) is slower

than the true kon (see Section 7.2). A second, related problem is that, following dissociation of the

analyte, it can rebind to the unoccupied ligand before diffusing out of the matrix and being washed

from the flow cell. Consequently, the measured dissociation rate constant (apparent koff) is slower

than the true koff (see Section 7.2). Although the dextran matrix may exaggerate these kinetic

artefacts (mass transport limitations and re-binding) they can affect all surface-binding techniques .

2.2. Applications

This section outlines the applications for which SPR is particularly well suited. Also described are

some applications for which it is probably not the technique of choice. Of course, future technical

improvements are likely to extend the range of applications for which the SPR is useful.

2.2.1. What SPR is good for

2.2.1.1. Evaluation of macromolecules

Most laboratories studying biological problems at the molecular or cellular level need to produce

recombinant proteins. It is important to be able to show that the recombinant protein has the same

structure as its native counterpart. With the possible exception of enzymes, this is most easily done

by confirming that the protein binds its natural ligands. Because such interactions involve multiple

residues, which are usually far apart in the primary amino acid sequence, they require a correctly

folded protein. In the absence of natural ligands monoclonal antibodies (mAbs) that are known to

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bind to the native protein are an excellent means of assessing the structural integrity of the

recombinant protein. The BIAcore is particularly well suited to evaluating the binding of

recombinant proteins to natural ligands and mAbs. Setting up an assay for any particular protein is

very fast, and the data provided are highly informative.

2.2.1.2. Equilibrium measurements (affinity and enthalpy).

Equilibrium analysis requires multiple sequential injections of analyte at different concentrations

(and at different temperatures). Because this is very time-consuming it is only practical to perform

equilibrium analysis on interactions that attain equilibrium within about 30 min. The time it takes to

reach equilibrium is determined primarily by the dissociation rate constant or koff; a useful rule of

thumb is that an interaction should reach 99% of the equilibrium level within 4.6/koff seconds. High

affinity interactions (KD < 10 nM) usually have very slow koff values and are therefore unsuitable for

equilibrium analysis. Conversely, very weak interactions (KD >100 µM) are easily studied. The small

sample volumes required for BIAcore injections (<20 µL) make it feasible to inject the very high

concentrations (>500 µM) of protein required to saturate low affinity interactions (11).

Equilibrium affinity measurements on the BIAcore are highly reproducible. This feature and the very

precise temperature control makes it possible to estimate binding enthalpy by van’t Hoff analysis

(12). This involves measuring the (often small) change in affinity with temperature (Section 7.4).

Although not as rigorous as calorimetry, much less protein is required.

2.2.1.3. Kinetic measurements

The fact that the BIAcore generates real-time binding data makes it well suited to the analysis of

binding kinetics. There are, however, important limitations to kinetic analysis. Largely because of

mass transport limitations it is difficult to measure accurately kon values faster than about 106 M-1s-1.

This upper limit is dependent on the size of the analyte. Faster kon values can be measured with

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analytes with a greater molecular mass. This is because the larger signal produced by a large analyte

allows the experiment to be performed at lower ligand densities, and lower ligand densities require

lower rates of mass transport. For different reasons measuring koff values slower than 10-5 s-1 or faster

than ~1 s-1 is difficult. Because the BIAcore is easy to use and the analysis software is user-friendly,

it is deceptively easy to generate kinetic data. However obtaining accurate kinetic data is a very

demanding and time-consuming task, and requires a thorough understanding of binding kinetics and

the potential sources of artefact (Section 7.2).

2.2.1.4. Analysis of mutant proteins

It is possible using BIAcore to visualise the capture of proteins from crude mixtures onto the sensor

surface. This is very convenient for analysing mutants generated by site-directed mutagenesis

(13,14). Mutants can be expressed as tagged proteins by transient transfection and then captured from

crude tissue culture supernatant using an antibody to the tag, thus effectively purifying the mutant

protein on the sensor surface. It is then simple to evaluate the effect of the mutation on the binding

properties (affinity, kinetics, and even thermodynamics) of the immobilised protein. This provides

the only practical way of quantifying the effect of mutations on the thermodynamics and kinetics of

weak protein/ligand interactions (15).

2.2.2. What SPR is not good for.

2.2.2.1. High throughput assays.

The fact the BIAcore can only sample can be analysed at a time, with each analysis taking 5-15 min,

means that it is neither practical nor efficient for high throughput assays. Automation does not solve

this problem because the sensor surface deteriorates over time and with re-use. Blockages or air

bubbles in the microfluidic system are also common in long experiments, especially when many

samples are injected.

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2.2.2.2. Concentration assays.

The BIAcore is also unsuitable for concentration measurements, because these require the analysis of

many samples in parallel, including the standard curve. A second problem is that, for optimal

sensitivity, concentration assays require long equilibration periods.

2.2.2.3. Studying small analytes.

Because the SPR measures the mass of material binding to the sensor surface, very small analytes

(Mr <1000) give very small responses. The recent improvements in signal to noise ratio have made it

possible to measure binding of such small analytes. However a very high surface concentration of

active immobilised ligand (~1 mM) is needed, and this is difficult to achieve. Furthermore, at such

high ligand densities accurate kinetic analysis is not possible because of mass-transport limitations

and re-binding (Section 7.2). Thus only equilibrium analysis is possible with very small analytes,

and then only under optimal conditions. This assessment may need to be revised as and when future

improvements are made in the signal to noise ratio.

3. General principles of BIAcore experiments

3.1. A typical experiment

A typical SPR experiment involves several discrete tasks.

• Prepare ligand and analyte.

• Select and insert a suitable sensor chip.

• Immobilise the ligand and a control ligand to sensor surfaces.

• Inject analyte and a control analyte over sensor surfaces and record response.

• Regenerate surfaces if necessary.

• Analyse data.

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While the ligand and analyte could be almost any type of molecule, they are usually both proteins.

This chapter focuses on the analysis of protein-protein interactions.

3.2. Preparation of materials and buffers

BIAcore experiments are frequently disrupted by small air bubbles or other particles passing through

the flow system. Usually these can be flushed out and the experiment repeated, wasting only time and

reagents. Occasionally, however, the damage is irreversible necessitating the replacement of an

expensive sensor chip or integrated fluidic cartridge. This can be minimised by following a few

simple rules (Section 9.2.1).

3.3. Monitoring the Dips

The output from the photo-detector array that is used to determine the surface plasmon resonance

angle (θspr, Section 9.1) can be viewed directly as 'dips'. The current BIAcore documentation does not

describe how to view and interpret dips, and so this information is supplied here (Protocol 1).

Protocol 1. Normal and abnormal 'dips'

Viewing the dips

1. Enter the service mode on the BIAcore control software by simultaneously pressing the control, alt, and s keys.

2. When the dialog box appears requesting a password ignore this and press the enter or return key. An additional Service menu will appear on the menu bar.

3. Select View dips from this menu. 4. A graph appears similar to the one in Figure 1 showing the amplitude of light reflected off the

sensor surface (Reflectance) measured over a small range of angles. The angle of the minimum reflectance ( θspr) is calculated by fitting a curve to all this data, thereby enabling θspr to be measured at a far greater resolution than the resolution at which the data are actually collected.

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Normal dip

The important feature of a normal dip is its depth (Figure 1, Dip1). Generally it bottoms out at a Reflectance of ~10000. When the refractive index above the sensor surface increases (e.g. because of protein binding), the dip shifts to the right while maintaining its shape and depth and (Dip 2).

Abnormal dip

There are two main types of abnormal dip. 1. The shallow dip (Dip 3). The θspr is measured along a section of the sensor surface rather than at

a single point. Refractive index heterogeneity on the surface gives heterogenous θspr values. When averaged the result is a shallow dip that does not reach below 10000 RUs. Heterogeneity can be the result of differences in the amount of material immobilised along the surface, in which case the dip is usually slightly shallow, or the result of small air bubbles or particles in the flow-cell, in which case the dip is very shallow.

2. No dip (Dip 4). A large change in the refractive index beyond the instrument dynamic range (Table 1) will shift the θspr so much that no dip is evident. Usually this is the result of air in the flow cell.

While slight shallowness of the dip is acceptable, and is common after coupling large amounts of protein, more severe abnormalities should not be ignored. Attempts should be made to return dips to normal by flushing the flow cells with buffer and/or regenerating the sensor surfaces. If this is ineffective a new sensor surface should be used.

4. Ligand

4.1. Direct versus indirect immobilisation

The most challenging step when setting up SPR experiments is immobilising of one of the proteins

(the ligand) to the sensor surface without disrupting its activity. Immobilisation can either be direct,

by covalent coupling, or indirect, through capture by a covalently coupled molecule.

The major advantage of direct covalent immobilisation is that it can be used for any protein provided

that it is reasonably pure (>50%) and has a pI > 3.5. However, it has three important drawbacks.

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Firstly, because they usually have multiple copies of the functional group that mediates

immobilization, proteins are coupled heterogeneously and sometimes at multiple sites. Secondly,

direct coupling often decreases or completely abrogates binding to analyte. And thirdly, directly-

coupled proteins are difficult to regenerate (see below).

Indirect immobilisation has the disadvantage that it can only be used for proteins that have a suitable

binding site or tag for the covalently coupled molecule. However, it has four important advantages,

which make it the method of choice in most cases. Firstly, proteins are seldom inactivated by indirect

coupling. Secondly, the protein need not be pure. It can be captured from a 'crude' sample. Thirdly,

all the molecules are immobilised in a known and consistent orientation on the surface. Finally, using

appropriate buffers it is often possible to dissociate selectively the non-covalent ligand/analyte bond,

thereby enabling the ligand surface to be re-used – a process termed ‘regeneration’.

4.2. Covalent immobilisation

4.2.1. A general approach

Instead of replicating the detailed protocols in the BIAcore literature (see Section 9.3), I will suggest

a general approach (Protocol 2) to covalent coupling, discussing several aspects in detail.

Protocol 2. An approach to covalent coupling of a protein 1. Select the coupling chemistry (Section 4.2.2). 2. Prepare the protein (Section 4.2.3). 3. Optimize the pre-concentration step (Section 4.2.4). 4. Couple the protein (Section 4.2.5). 5. Evaluate the activity of the immobilised protein (Section 4.5). 6. Establish conditions for regeneration (Section 4.2.6). 7. Adjust the immobilization conditions (Section 4.2.7).

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4.2.2. Choice of chemistry

There are three main types of coupling chemistry, which utilize, respectively, amine (e.g. lysine),

thiol (cysteine) or aldehyde (carbohydrate) functional groups on glycoproteins (Table 3). All

covalent coupling methods utilize free carboxymethyl groups on the sensor chip surface. They can

therefore be used for any of the sensor chips that have such carboxymethyl groups (See Table 2). If

the protein to be immobilised has a surface-exposed disulphide or a free cysteine, ligand-thiol

coupling is probably the method of choice. Failing this, amine coupling should be tried in the first

instance. If amine coupling inactivates the protein (as assessed by ligand and/or mAb binding),

aldehyde coupling can be attempted, provided that the protein is glycosylated. Detailed protocols are

available from BIAcore for all coupling techiques (see Section 9.3). Only amine coupling is

described here in some detail.

4.2.3. Prepare the protein

Only a modest amount (5-10 µg) of protein is needed. The major requirement is that the protein is

pure and has a high level of activity. Because direct coupling is relatively indiscriminate, all protein

in the preparation will be coupled. Thus if the preparation is contaminated by other proteins or is

partially active, the level of binding observed will be proportionally decreased. Because the protein

needs to be diluted into the pre-concentration buffer (Section 4.2.4) to a final concentration of 20-50

µg/ml, the stock should be fairly concentrated (> 0.5 mg/ml). If the protein is in solutions that are

strongly buffered or contain high salt concentrations, primary amine groups or sodium azide, then

these must either be dialysed out or much larger dilutions (1:100) made. In the latter case the protein

will need to be at higher concentrations (>2 mg/mL).

4.2.4. Pre-concentration

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The purpose of pre-concentration is to concentrate the protein to very high levels (>100 mg/mL)

within the dextran matrix, thereby driving the coupling reaction. Without pre-concentration far

higher concentrations of protein would need to be injected to get equivalent levels of coupling. Pre-

concentration is driven by an electrostatic interaction between the negatively charged carboxylated

dextran matrix and positively charged protein. To this end the protein is diluted into a buffer with a

low ionic strength (to minimize charge screening) with a pH below its isoelectric point or pI (to give

the protein net positive charge). However amine coupling is most efficient at a high pH because

activated carboxyl groups react better with uncharged amino groups. Thus the highest pH compatible

with pre-concentration is determined empirically (Protocol 3). Electrostatically-bound protein should

dissociate rapidly and completely when injection of running buffer resumes, both because the

proteins net positive charge will decrease and because electrostatic interactions will be screened by

the high ionic strength of the running buffer. Incomplete dissociation suggests that the interaction

was not purely electrostatic, perhaps because of the binding, at low pH, of a denatured form of the

protein.

Protocol 3. Determining the optimum pre-concentration conditions for a protein 1. Dilute the protein to a final concentration of 20-50 µg/mL (final volume 100 µL) into pH 6.0,

5.5, 5.0, 4.5 and 4.0 pre-concentration buffers (see Section 9.2.2). 2. Start a manual BIAcore run using a single flow cell (flow-rate 10 µL/min) 3. Inject 30 µL of each sample, beginning at pH 6.0 and working down. 4. If no electrostatic interaction is observed continue with protein samples diluted in pH 3.5 and 3.0

pre-concentration buffers. 5. Use the highest pH at which >10000 RU of protein binds electrostatically during the injections. 6. Check that all the bound protein dissociates after the injection. If not it suggests that not all the

protein was bound electrostatically and that the protein denatures irreversibly at that pH.

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4.2.5. Amine coupling

There is a standard protocol for amine coupling (Protocol 4). The first step is to activate the

carboxymethyl groups with N-hydroxysuccinimide (NHS), thus creating a highly reactive

succinimide ester which reacts with amine and other nucleophilic groups on proteins. The second

(coupling) step is to inject the protein in pre-concentration buffer, thereby achieved high protein

concentrations and driving the coupling reaction. The third (blocking) step, blocks the remaining

activated carboxymethyl groups by injecting very high concentrations of ethanolamine. The high

concentration of ethanolamine also helps to elute any non-covalent bound material. The final

regeneration step is optional. When a protein is coupled for the first time it is advisable not to include

any regeneration step. If it is included and the protein has poor activity it will not be clear whether

covalent coupling or regeneration was responsible for disrupting the protein. The structural integrity

of the protein should be evaluated (Section 4.2.6) before regeneration is attempted. Once

regeneration conditions have been established these can be added on to the coupling protocol.

Protocol 4. Amine coupling

Reagents

120 µL of protein at 20-50 µg/ml in suitable pre-concentration buffer (Protocol 3) 120 µL of EDC (0.4 M) mixed 1:1 with NHS (0.1 M). Make up just before coupling 120 µL of ethanolamine/HCl 1 M, pH 8.0 120 µL of regeneration solution (if regeneration conditions known)

Procedure

1. Establish pre-concentration conditions (Protocol 3) 2. Set flow-rate to 10 µL/min

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3. Activation: inject 70 µL of EDC/NHS 4. Coupling: inject 70 µL of protein 5. Blocking: inject 70 µL of ethanolamine 6. Regeneration: inject 30 µL of regeneration solution (if regeneration conditions known, see

Protocol 5). 7. View the 'dips' (Protocol 1) to confirm that the immobilisation is homogeneous.

4.2.6. Regeneration

Once a covalently immobilised protein has been shown to be active with respect to binding its natural

ligand or a monoclonal antibody (Section 4.4), regeneration can be attempted. A general approach to

establishing regeneration conditions can be used (Protocol 5). The goal here is to elute any non-

covalently bound analyte without disrupting the activity of the ligand. Regeneration allows surfaces

to be re-used many times, saving both time and money. However establishing ideal regeneration

conditions can be a very time-consuming, and in many cases impossible, task. Thus it may be more

cost-effective to opt for imperfect or no regeneration, using new sensor surfaces instead.

Protocol 5. Establishing regeneration conditions

Regeneration solutions

• These fall into four main classes: divalent cation chelator, high ionic strength, low pH, high pH (Section 9.2.2).

• If the interaction is likely to be dependent on divalent cations try buffers with EDTA. • If the monomeric interaction is weak, try high ionic strength buffers. • Otherwise start with a low pH buffer. • If this is without effect, try high pH buffer.

Approach

1. Covalently couple ligand to the surface. 2. Make up enough analyte for several injections.

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3. Inject analyte over the immobilised ligand and measure the amount of binding. 4. Inject selected regeneration buffer for 3 min, and measure the amount of analyte that remains

bound after this. 5. If the decrease is <30% switch to a different class of regeneration buffer and return to step 4. 6. If the decrease is >30% but <90%, try repeated injections. If this fails to elute >90% select a

stronger regeneration buffer of the same type and return to step 4. 7. If this fails, try a different class of buffer and return to step 4. 8. When >90% of bound analyte is eluted, return to step 3, using identical analyte and injection

conditions. If binding post-regeneration remains >90% of binding before regeneration, the regeneration conditions may be adequate.

9. If residual activity is <90%, select a different class of regeneration buffer. If residual binding is very low it may be necessary to return to step 1, starting with a new surface.

10. If two different types of buffer give partial elution, try using both sequentially.

4.2.7. Adjusting the immobilisation conditions.

Since the level of ligand coupling achieved is unpredictable it is usually necessary to modify the

initial protocol in order to achieve the desired immobilization level. It is also often necessary to

create several surfaces on the same chip with different levels of coupling. The best way to achieve

different levels of coupling is to change the duration of the activation step, by varying the volume of

NHS/EDC injected. The level of coupled ligand varies in proportion with the duration of the

activation step. Thus, a two-fold reduction in activation period will usually lead to a two-fold

reduction in coupling. It is possible using an option in the inject command to couple ligand

simultaneously in multiple flow cells, varying only the length of activation step.

4.3. Non-covalent immobilisation (ligand capture)

There are two requirements for non-covalent or indirect immobilization of a ligand (henceforth called

'ligand capture'). First, it must be possible to obtain or create a sensor surface that can capture a

ligand. Ideally, it should be possible to regenerate this surface so that repeated capture is possible.

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Second, the ligand needs to have a suitable binding site or modification that allows it to be captured.

4.3.1. Using an existing strategy

A number of established techniques are available for ligand capture (Table 4). When expressing a

recombinant protein to be used in SPR studies it is advisable to consider adding a suitable domain or

peptide motif so that one of these techniques can be used. The precise choice of tag will depend on

whether the tag is needed for purification and what other uses are envisaged for the ligand.

• If it is envisaged that the ligand is to be used as an analyte in SPR studies it should be

monovalent or, if multivalent (Fc- and GST-chimeras), the tag should be readily removable.

• If the ligand is to be used for structural studies, the tag should also be removable.

• If the ligand is to be used to probe for binding partners on cells or tissues it should be multivalent,

or it should be possible to make it multivalent.

• If the ligand is to be captured from crude mixtures (i.e. after expression without purification) the

capturing agent needs to have a high affinity and to be highly specific. For example, CD4 (14)

and anti-human IgG1 (13) mAbs have been shown to be suitable for this purpose.

4.3.2. Developing a new strategy

The widespread availability of purified monoclonal or polyclonal mAbs, and their ability to tolerate

harsh regeneration conditions, make them suitable reagents for non-covalent immobilization.

Typically there is a need to immobilize a number of related ligands, with an invariant and a variant

portion. If several antibodies against the invariant portion are available, it is likely that at least one of

these will be suitable for indirect coupling. A basic approach to developing such a method is

described in Protocol 6.

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Protocol 6. Developing a new antibody-mediated indirect coupling method 1. Obtain a panel of antibodies that can bind a suitable ligand and are available in pure form. 2. Analyse the binding properties and select a high affinity antibody with a slow koff. 3. Covalently couple the antibody by amine coupling (Protocol 4) and check for activity. If inactive,

try a different antibody. 4. Establish regeneration conditions (Protocol 5). 5. If suitable regeneration conditions cannot be found, try a different antibody.

4.4. Activity of immobilised ligand

It is important to evaluate the functional integrity of the immobilised protein. This is best achieved by

using a protein which binds to correctly-folded ligand. An ability to bind its natural ligand is

reassuring evidence that an immobilised ligand is functionally intact. Since monoclonal antibodies

(mAbs) usually bind to 'discontinuous epitopes' on a protein, they are excellent probes of protein

structure. It is preferable to check the binding of several mAbs, including ones that bind to the same

binding sites or, failing this, the same domain of the natural ligand. MAbs are particularly useful if

the natural binding partner has not been identified and/or a candidate binding partner being assessed

for an interaction with the ligand. In is important to know not just whether immobilised ligand is

'active' but also what proportion is active (Protocol 7).

Protocol 7. Quantitating binding levels

The ligand activity (ActL) , stoichiometry (S), molecular mass of ligand (ML) and analyte (MA), and the analyte binding level at saturation (A) are related as follows:

LA

MMSActL

A

L ∗

=∗

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Where L is the level of ligand immobilised S is the molar ratio of analyte to ligand in analyte/ligand complex

The product ActL*S is readily calculated once A and L have been measured. Either ActL or S need to be independently determined in order to calculate the other. A convenient way to measure ActL by SPR is to use Fab fragments of mAbs specific for the ligand. Intact mAbs are less useful because of the uncertaintly as to their binding stoichiometry.

4.5. Control surfaces

A control surface should be generated which is as similar as possible to the ligand surface, including

similar levels of immobilisation. This is to measure non-specific binding and to record the

background response. The immobilisation levels need to be similar because this affects the

background response measured with analytes that have a high refractive index (Section 5.3).

4.6. Re-using sensor chips

Because each sensor chip has several flow cells, it is common to have unused flow cells at the end of

an experiment. In addition many covalently coupled ligands are very stable, enabling surfaces to be

re-used over several days. It is therefore convenient to be able to remove and reinsert sensor chips in

the BIAcore (Protocol 8).

Protocol 8. Reusing sensor chips • Undock the sensor chip, choosing the empty flow cell option in the dialogue box. • Store the sensor chip in its cassette at 4ºC. • When it is to be re-used, re-insert and dock the sensor chip. • After priming the system check the 'dips' (Section 3.3).

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5. Analyte

The extent to which the analyte needs to be characterised depends on the nature of the experiment.

Quantitative measurements (Section 7) require that the analyte is very well characterised and of the

highest quality. In contrast, this is less important for qualitative measurements (Section 6).

5.1. Purity, activity and concentration

In order to determine affinity and association rate constants it is essential that the concentration of

the injected material is known with great precision. The only sufficiently accurate means of

measuring concentration is to use a spectrophometer to measure the optical density of a solution of

pure protein, usually at 280 nm (OD280). In order to calculate the concentration from the absorption

at OD280, two additional measurements are required. Firstly, it is necessary to determine the

extinction coefficient. Although this can be calculated from the primary sequence it is best

determined directly by amino acid analysis of a sample of the protein with a known OD280. Secondly,

it is important to assess what proportion of the purified protein is 'active', i.e. able to bind to the

ligand. This can be done by depletion experiments in which the ligand-coated sepharose beads are

used to deplete the analyte from solution (16). mAb-coated sepharose beads can be used instead if

ligand-coated beads are impractical. If all the analyte can be depleted in this way it is 100% active.

5.2. Valency

In general, affinity and kinetic measurement require that each analyte molecule has a single binding

site, i.e. is monovalent. If the protein has a single binding site it is only necessary to show that it

exists as a monomer in solution. This is most readily achieved by size-exclusion chromatography

(Chapter 3) or analytical ultracentrifugation (Chapters 4 and 5). It is important to emphasise that

- 20 -

these analytical techniques will not detect the presence of very low concentrations of multivalent

aggregated material (17,18). In order to ensure that this material does not contribute to the binding it

is ESSENTIAL to purify the monomeric peak by size exclusion chromatography immediately before

BIAcore experiments and to analyse it before concentrating or freezing it. Only when it has been

shown that concentration, storage or freezing do not affect the measured affinity and kinetic

constants is it wise to deviate from this strict principle. Fortunately the presence of multivalent

aggregates is readily excluded by analysis of the binding kinetics (Section 7.2). If the dissociation of

bound analyte is monophasic (mono-exponential) multivalent binding can be ruled out. If

dissociation is bi-exponential with >10 fold difference in the two koff values, multivalent binding is

likely. Bi-exponential dissociation with smaller differences in the two koff values could have several

explanations (see Section 7.2).

5.3. Refractive index effect and control analytes

When an analyte is injected over a surface it is important, for two reasons, to perform a second

injection with a control analyte. Firstly, this helps rule out non-specific binding. Secondly, it controls

for any refractive index artefacts. These occur when the background signal measured during the

injection of an analyte sample differs between flow cells. Clearly such an artefact will create

problems for affinity measurements. It can occur whenever there is a substantial difference between

surfaces. For example, if very different levels of material are immobilised on each surface. In this

case, because the immobilised material displaces volume, the volume accessible to the injected

analyte sample will differ between flow-cells. If the analyte sample has a higher refractive index than

the running buffer, a larger background signal will be seen from the surface with less immobilised

material. This artefact is greater when (i) there are big differences in the levels of immobilised

material (e.g. >2000 RUs), (ii) the background signal is very large (e.g. >1000 RUs), and (iii) the

- 21 -

binding response is much smaller (<10%) than the background response. These conditions are

common when measuring very weak interactions, because high concentrations of analyte are

injected, and with low molecular weight analytes, which give a very small response. A refractive

index artefact can be detected by injecting a control solution with a similar refractive index to the

analyte sample. If the control solution gives the same response in both flow-cells a refractive index

artefact can be excluded. Refractive index artefacts are most easily avoided by taking care to

immobilise the same amount of total material on both the control and ligand sensor surfaces.

5.4. Low molecular weight analytes

Recent improvements in the signal to noise ratios of BIAcore instruments have enabled binding to be

detected of analytes with Mr as low as 180 (19). There are two major problems associated with such

studies. Firstly, very high densities of ligand must be immobilised in order to detect binding. The

levels can be calculated using equation in Protocol 7. For an analyte of Mr ~200 that binds a ligand

with an Mr of ~40000, approximately 10000 RU of active ligand needs to be immobilised to see 50

RU of analyte binding. Achieving this level of immobilization is very difficult. A second problem is

that with such small binding responses refractive index effects become significant. The latter can be

avoided by dissolving and/or diluting the analyte in the running buffer and using a control flow cell

with very similar levels of immobilization.

6. Qualititative analysis; do they interact?

6.1. Positive and negative controls

The main purpose of a qualitative analysis is to establish whether or not there is an interaction

between a given analyte and ligand. If binding is detected it is necessary to test negative controls to

exclude a false positive. These include negative ligand controls and negative analyte controls.

- 22 -

Blocking experiments, using molecules known to block the interaction, are also useful negative

controls. In contrast, if no binding is detected it becomes necessary to run positive controls to

establish whether this reflects the absence of an interaction, a very low affinity, or an artefact

resulting from defective ligand or analyte. The ligand is readily assessed by showing that it can bind

to one or more mAbs or additional analytes should they exist. The analyte can be tested by injecting

it over a surface with either a known interacting ligand or or mAb immobilised to the surface.

6.2. Qualitative comparisons using a multivalent analyte

The binding of a multivalent analyte can be heavily influenced by the level of immobilised ligand,

since the latter will influence the valency of binding. Thus when comparing the binding of a

multivalent analyte to different ligands it is important that these ligands are immobilised at

comparable surface densities.

7. Quantitative measurements

Quantitative measurements are far more demanding than qualitative measurements because of the

quality and amount of materials required and the difficulties associated with designing the

experiments and analysing the data. When undertaking these measurements it is particularly

important to understand the various pitfalls and how these can be avoided (4,5,8). Any quantitative

analysis on the BIAcore requires that the analyte binds in a monovalent manner. Since many binding

parameters are temperature dependent it is important to perform key measurements at physiological

temperatures (i.e. 37°C in mammals). Considering how easy it is to regulate the temperature on the

BIAcore it is surprising how seldom this is done.

- 23 -

7.1. Affinity

7.1.1. Concepts

As has been described earlier in this volume (see, e.g. Chapters 1, 3-5), there are a number of ways to

represent the affinity of an interaction.

• The ‘association constant’ (KA) or affinity constant is simply the ratio at equilibrium of the

‘product’ and ‘reactant’ concentrations. Thus for the interaction A + B ↔ AB

BA

ABA CC

CK∗

=

Note that KA has units M-1 (i.e. L.mol-1)

• Many prefer to express affinity as the ‘dissociation constant’ or KD, which is simply the inverse

of the KA, and therefore has the units M.

• Affinity can also be expressed as the binding energy or, more correctly, the standard state molar

free energy (∆G°). This can be calculated from the dissociation constant as follows.

oD

CKRTG ln=°∆

where T is the absolute temperature in Kelvin (298.15 K = 25 °C)

R is the Universal Gas Constant (1.987 cal.K-1.mol-1)

Co is the standard state concentration (i.e. 1 M)

7.1.2. Experimental design

In principle the affinity constant can be measured directly by equilibrium binding analysis, or

calculated from the kon and koff. However, because of the difficulties associated with obtaining

definitive kinetic data on the BIAcore, equilibrium binding analysis is more reliable. It involves

injecting a series of analyte concentrations and measuring the level of binding at equilibrium. The

- 24 -

relationship between the binding level and analyte concentration enables the affinity constant to be

calculated (7). A basic approach to such measurements is outlined on Protocol 9.

7.1.2.1. Preliminary steps

Because equilibrium measurements are unaffected by mass-transport or re-binding artefacts, high

levels of immobilisation can be used to increase the binding response. This is particularly useful

when the background signal is high or the analyte very small.

Ideally, equilibrium affinity measurements require that the level of active ligand on the surface is the

same for each concentration of analyte injected. This is usually straightforward when the ligand is

covalently coupled (and so does not dissociate) and the analyte dissociates spontaneously within a

few minutes (so that regeneration is not required). In cases where regeneration is required it must be

shown that ligand activity is unaffected by repeated regeneration. Where captured ligands dissociate

spontaneously or require regeneration, it may be difficult to maintain the level of the ligand constant.

It is possible to correct for this if the level of active ligand can be accurately monitored (20).

It is important to ensure that the analyte injections reach equilibrium. While the approximate time it

takes to reach equilibrium can be calculated (see footnote to Protocol 9), it is advisable to measure

this directly in preliminary experiments under the same conditions (flow rate, analyte concentration,

ligand density) as those to be used for the affinity measurements. Enough time must be allowed

following the injection for the bound analyte to dissociate completely from the sensor surface (see

footnot to Protocol 9). If dissociation is incomplete, or takes too long, it may be necessary to enhance

dissociation by injecting regeneration solution.

7.1.2.2. The experiment

Ideally the analyte concentration should be varied over four orders of magnitude, from 0.01* KD to

100 * KD. However it is often only practical to vary the concentration over 2-3 orders of magnitude.

- 25 -

This can be achieved with 10 two-fold dilutions starting at between 10 * KD and 100 * KD.

An assumption in these affinity measurements is that the level of active immobilised ligand remains

constant. This should be checked by showing that a reference analyte binds to the same level at the

beginning and end of the experiment. A second internal control is to reverse the order of injections.

An efficient way of doing this is to work up from the lowest concentration of analyte, give one

injection at the highest concentration, and then work back down to the lowest concentration. The

same affinity should be obtained irrespective of the order of injections.

7.1.3. Data analysis

In order to derive an affinity constant from the data a particular binding model must be used. The

simplest (Langmuir) model (A+L↔AL) is applicable in the vast majority of cases. It assumes that

the analyte (A) is both monovalent and homogenous, that the ligand (L) is homogeneous, and that all

binding events are independent. Under these conditions data should conform to the Langmuir binding

isotherm,

DA

A

KCMaxCBound

+=

*

where "Bound" is measured in RUs and "Max" is the maximum response (RUs).

CA is the concentration of injected analyte and KD is in the same units as CA (normally

M)

The KD and Max values are best obtained by non-linear curve fitting of the equation to the data using

a suitable computer software such as Origin (MicroCal) or Sigmaplot.

A Scatchard plot of the same data (see Chapter 3), obtained by plotting Bound/CA against Bound, is

useful for visualising the extent to which the data conform to the Langmuir model. A linear

- 26 -

Scatchard plot is consistent with the model. Scatchard plots alone should not be used to estimate

affinity constants since they place inappropriate weighting on the data obtained with the lowest

concentrations of analyte, which are generally the least reliable.

Non-linear Scatchard plots indicate that the data do not fit the Langmuir model. Before considering

models that are more complex it is important to exclude trivial explanations (Section 7.1.5).

7.1.4. Controls

Several artefacts can result in erroneous affinity constants. These include an effect of ligand

immobilisation on the binding, an error estimating the active concentration of analyte, and an

incorrect assumption that the analyte is monovalent. The most rigorous control is to confirm the

affinity constant in the reverse or 'upside down' orientation since this excludes all three artefacts. If

this is not possible the experiment should be repeated with the ligand immobilised in a different way,

which addresses the possible effects of immobilization on binding. The affinity should be also be

confirmed with two independently-produced batches of protein and with different recombinant forms

of the same proteins.

7.1.5. Non-linear Scatchard Plots

A non-linear Scatchard plot indicates that binding does not conform to the Langmuir model. Many

binding models can be invoked to explain non-linear Scatchard plots. Distinguishing between these

models can be very difficult and is beyond the scope of this Chapter. The priority should be to

exclude trivial explanations for non-linear Scatchard plots. A 'concave up' Scatchard plot is the most

common deviation from linearity. It may be a consequence of heterogeneous ligand, multivalent

analyte, or (rarely) negative cooperativity between binding sites. A trivial cause of analyte

heterogeneity is the presence of multivalent analyte. Ligand heterogeneity may be a consequence of

- 27 -

immobilisation. A 'concave down' Scatchard plot is unusual and indicates either positive

cooperativity between binding sites or self-association of the analyte, either in solution or on the

sensor surface.

The control experiments outlined in 6.1.2.4 will help to eliminate some trivial explanations. For

example, if the analyte has a multivalent component the non-linear Scatchard plot will not be evident

in the reverse orientation. The shape of the Scatchard plot will also depend on the surface density of

ligand. If the ligand immobilization is responsible for heterogeneity, this should be eliminated in the

reverse orientation and if the ligand is immobilised indirectly.

Protocol 9. Affinity measurements

Preliminary steps

1. Immobilise the ligand and a control. High levels of immobilisation are acceptable. 2. Ensure that the analyte is monomeric and binds monovalently. Determine accurately the

concentration of the analyte and the proportion that is active. 3. Determine the time it takes to reach equilibrium and the time it takes for the bound analyte to

dissociate completely from the sensor surface. While this should be done empirically, under the conditions to be used for the equilibrium measurements, approximate times can be calculated from the koff

a. 4. If necessary, establish regeneration conditions (Protocol 5). 5. Obtain a rough estimate of the KD by injecting a series of five-fold dilutions.

Measurements

1. Prepare a dilution series of analyte starting at 10-100 times the KD with at least 9 two-fold dilutions thereof. There should be enough for two injections at each concentration except the highest concentration, where only enough for one injection is required. A minimum of 17 µL of sample is required per injectionb.

2. Make up separate control sample of analyte (at concentration ~ KD) with enough for 2 injections. 3. Set the flow rate. To conserve sample this can be as slow as 1 µL.min-1.

- 28 -

4. Inject the control sample. 5. Inject the dilution series starting from low and moving up to the highest concentration (low-to-

high) and then moving back down to the low concentrations (high-to-low). Inject the highest concentration only once. It is important to inject for a period sufficiently long to reach equilibrium. Either enough time must be allowed for spontaneous dissociation or the analyte must be eluted with regeneration buffer.

6. Repeat the injection of the control sample. 7. For all injections measure the equilibrium response levels in the ligand and control flow cells.

The difference between these two is the amount of binding at each concentration.

Data analysis

1. Plot the binding versus the concentration for both the low-to-high and the high-to-low series (20).

2. Fit the Langmuir (1:1) binding isotherm to the data by non-linear curve fitting. Use this to

determine the KD and maximal level of binding.

3. Do a Scatchard plot and check if it is linear. The points on the plot where binding is less than 5%

of maximum are highly inaccurate and should be ignored.

4. If the Scatchard plot is not linear do further experiments to establish cause (Section 7.1.5).

Controls

1. The affinity constant should be confirmed in the reverse orientation. 2. Should this be impossible (e.g. because the ligand is multivalent) the affinity constant should be

confirmed with the ligand immobilised by a different mechanism, preferably by ligand capture. 3. Use at least two independently produced batches of protein. 4. Use different recombinant forms of the same proteins. aBoth the time taken to reach equilibrium and the time it takes for the bound analyte to dissociate are governed primarily by the koff. For the simple 1:1 model binding will reach 99% of the equilibrium level within 4.6/koff seconds. Similarly, it will take 4.6/koff seconds for 99% of the analyte to dissociate. Thus for koff ~0.02 s-1, equilibrium will be reached within ~ 230 s and the bound analyte will take ~230 s to dissociate. bIf the Quickinject command is used as little as 15 µL of analyte sample is used.

- 29 -

7.2. Kinetics

7.2.1. Concepts

The period during which analyte is being injected is termed the 'association phase' whereas the period

following the end of the injection is termed the 'dissociation phase'. During the association phase

there is simultaneous association and dissociation. Equilibrium is reached when the association rate

equals the dissociation rate. Under ideal experimental conditions only dissociation should take place

during the dissociation phase. In reality some re-binding (see below) often occurs. The main factors

affecting the association rate are the concentration of analyte near the ligand (CA), the concentration

of ligand (CL), and the association rate constant (kon). Because of the high surface density of ligand

on the sensor surface, the rate at which analyte binds ligand can exceed the rate at which it is

delivered to the surface (referred to as mass transport). In this situation binding is said to be mass

transport limited. Analysis of association rate under mass transport limited conditions will yield an

apparent kon that is slower than the true kon. It is difficult to determine the kon under these

circumstances, and so experimental conditions must be sought in which mass-transport is not

limiting. Analyte is transported to the surface by both convection and diffusion. Convection transport

can be increased simply by increasing the flow rate. However even at the maximal flow-rates

permissible mass transport can still be limiting (10,16) because there is an unstirred 'diffusion' layer

near the sensor surface through which transport is solely by diffusion (5). In this case mass transport

limits can only be avoided by decreasing the surface density of immobilised ligand.

The main factors affecting the analyte dissociation rate are the surface density of bound analyte, the

dissociation rate constant (koff), and the extent to which dissociated analyte rebinds to ligand before

leaving the sensor surface (termed 're-binding'). The latter is also a consequence of mass transport

deficiency but here it is transport away from the surface that is limiting. Convection transport can be

- 30 -

increased by increasing the flow rate. However re-binding will still occur because diffusion out of the

unstirred layer is little affected by convection transport. Re-binding is most easily avoided by

decreasing the level of ligand immobilised on the surface. An alternative method is to inject during

the dissociation phase a competing molecule that can rapidly bind to free analyte or ligand and block

re-binding (21). If it binds ligand the competing molecule needs to be small so that it does not

influence the SPR signal. Finally, when the ligand is saturated the initial part of the dissociation

phase will not be affected by re-binding, since no free ligand is available for re-binding. However

such selective analysis of a part of the dissociation phases should be avoided; it provides no

indication as to whether the data conforms to any particular binding model and can give highly

misleading results.

In summary, mass transport limitations, which lead to an underestimation of the intrinsic kinetics, are

aggravated by low flow rates, high levels of immobilised ligand, and high intrinsic association rate

constants. They can be reduced by increasing the flow rate and, most importantly, lowering the level

of immobilised ligand.

7.2.2. Experimental design

Because mass transport may limit binding it is essential to use the lowest density of ligand that gives

an adequate level of analyte binding. Depending on the background response 100 RU of binding

should be adequate. In order to determine whether binding is limited by mass transport the kinetics

should be measured in several flow cells with different levels of immobilised ligand. The

immobilization level should vary at least two-fold.

- 31 -

Protocol 10. Kinetic measurements

Preliminary steps

1. Immobilise the ligand in three flow cells at different levels (e.g. 500, 1000, and 2000 RU). 2. Immobilise a control ligand in the remaining flow cell at a level midway between the range of

immobilisation levels in the other three flow cells. 3. Ensure that the analyte is monomeric and binds monovalently. Determine accurately the

concentration of the analyte. 4. Determine the time it takes reach equilibrium and for the bound analyte to dissociate completely

from the sensor surface (see footnote in Protocol 9). If less than 4-5 seconds (fast) it will be necessary to collect at the maximal rate possible (10 Hz). This is only possible if data is collected from one flow cell at a time. Because the sample needs to be injected once for each flow cell studied, more sample is needed.

5. Establish regeneration conditions if necessary. With kinetic determinations it is not as important to maintain the same level of active ligand for each injection.

Measurements

1. Prepare a two-fold dilution series of the analyte ranging from concentrations of 8*KD to approximately 0.25*KD. Take care to prepare enough sample for the special kinetic injection command (KINJECT), which utilizes more material. If kinetics are fast and a high data collection rate is needed, enough analyte needs to be prepared for separate injections in each flow-cell.

2. Set the flow rate to 40-100 µL/min in order to maximize analyte mass transport. The duration of the injection is not critical since binding does not need to reach equilibrium. However, equilibrium should be approached at the higher analyte concentrations.

3. Inject the dilution series in any particular order. It is usual to start from lower concentrations.

Data analysis

1. Use the BIAevaluation software supplied by BIAcore. 2. Subtract the response in the control flow cell from the responses in each of the ligand flow cells. 3. Group and analyse together the binding curves obtained with each dilution series, one flow cell at

a time (with control responses subtracted). 4. If equilibrium is reached within 1 second the association phase will not produce useful data. In

this case only the dissociation phase should be analysed. 5. Attempt a global fit of the simple 1:1 binding model to the entire series of curves. Include in the

- 32 -

fit as much of the association and dissociation phase as possible. 6. Repeat the analysis with data obtained at the other levels of ligand immobilisation. In order to

prove that binding is not limited by mass transport it is necessary to show that the same rate constants are obtained at two different ligand immobilization levels. If this is not possible the measured rate constants should be considered to be lower limits of the true rate constants (16,18).

7. If poor fits are obtained using the simple 1:1 binding model, the binding is considered complex. After excluding trivial explanations (Table 5) an attempt should be made to establish the cause (Table 6).

Controls

1. The kinetics constants should always be confirmed in the reverse orientation and/or with the ligand immobilised in a completely different manner.

2. Always confirm the results using separate batches of recombinant protein. 3. If possible, confirm the results using different recombinant forms of the same protein.

A second important point is that more of the analyte is needed for kinetic analysis. This is because

the experiments are performed at a high flow-rate, the KINJECT command wastes more material,

and separate injections may be required for each flow-cell.

7.2.3. Data analysis

Analysis of kinetic data is best performed using the BIAevaluation software supplied with the

instruments as this has been designed especially for the purpose (9). Another programme CLAMP

(available at http://www.hci.utah.edu/cores/biacore/) has also been designed specifically for analysis

of kinetic data generated on the BIAcore (22). While a complete discussion of kinetic theory is

beyond the scope of this review a basic approach to kinetic analysis is provided instead. After

subtracting the background responses (obtained in the control flow-cells) an attempt should be made

to fit the simple 1:1 Langmuir binding model to the data. For any particular sensorgram as much of

the data as possible should be included in the fit. This normally includes the entire association and

- 33 -

dissociation phases, omitting only the 'noisy' few seconds at the beginning and end of the analyte

injection. Noise in the dissociation phase is reduced by using the KINJECT command. It is good

practise to fit both the association and dissociation phases simultaneously rather than separately.

However the association phase cannot be analysed if equilibrium is attained within 2-4 s, which is

usually the case if the koff is > 1 s-1. In contrast, the dissociation phase can analysed even if the koff is

>1 s-1 (11,20). A rigorous test of the binding model is to fit it simultaneously to multiple binding

curves obtained with different analyte concentrations. This global fitting (8,9) establishes whether a

single 'global' kon and koff provide a good fit to all the data. An important internal test of the validity

of the kinetic constants is to determine whether the calculated KD (KDcalc= koff/kon) is equal to the KD

determined by equilibrium analysis.

When a poor fit is obtained to the data using the simple 1:1 binding model the binding kinetics are

considered complex. Since the most likely explanation for this is experimental artefact, initial efforts

should be directed at excluding trivial causes (Table 5). Only when trivial explanations have been

excluded should any effort be expended on trying to establish what complex binding model explains

the kinetics. This is a difficult and often impossible task (8,9). The simple 1:1 binding model predicts

that both the association and dissociation phases are monoexponential, i.e. described by an equation

with a single exponential term. When a poor fit is obtained excellent fits can usually be obtained

using equations with two exponential terms (a bi-exponential fit). Because all complex binding

models generate equations with two or more exponential terms, it is usually impossible to distinguish

between different models by curve fitting alone. Instead further experiments need to be performed

(Table 6).

7.2.4. Controls

The same controls should be performed as for the affinity measurements (see Section 7.1.4 and

- 34 -

Protocol 10)

7.3. Stoichiometry

The binding stoichiometry can be determined if the molecular mass of ligand and analyte are known

and the activity of the ligand is known. The basic approach is to immobilise a defined amount of

ligand and then saturate this with analyte. The stoichiometry can then be calculated according to

Protocol 7. Because it is very difficult to saturate with analyte the maximum level of analyte binding

is best obtained by doing a standard equilibrium affinity determination. A fit of the simple 1:1

binding model to this data yields the maximum level of analyte binding as well as a KD. The key

problem is establishing the activity of the immobilised ligand. If the ligand has 100% activity in

solution and is immobilised by ligand capture, it is reasonable to assume that it is all active. Activity

levels can also be determined using an Fab fragment of a mAb specific for the ligand. Finally, the

stoichiometry should be identical when measured in the reverse orientation.

7.4. Thermodynamics

The binding energy or affinity includes contributions from changes in enthalpy (heat absorbed or

∆H) and entropy (increased disorder or ∆S).

∆G = ∆H - T*∆S

While ∆S cannot be measured, ∆H (or the heat absorbed upon binding) can be measured directly, by

microcalorimetry (∆Hcal), or indirectly, by van't Hoff analysis (∆HvH). If it is assumed that ∆H and

∆S° are temperature-independent, the linear form of the van’t Hoff equation can be used.

RS

TRH

CK vH

oD °∆

−∗

∆=ln

where Co is the standard state concentration (1 M) and ∆S° is the change in entropy in

the standard state. KD is measured over a range of temperatures and ln(KD/Co) plotted against 1/T. If - 35 -

linear, the slope of this plot equals ∆HvH/R. A drawback of this approach is that ∆H varies with

temperature for protein/ligand interactions and so the plot is not linear. Consequently, KD needs to be

measured over a small range around the temperature of interest, and the slope determined within this

range. This is technically difficult and likely to be inaccurate. A more rigorous approach is to

measure the affinity (∆Gº, see section 7.1.1) over a wider range of temperatures and then fit an

integrated (non-linear) form of the van't Hoff equation to the data (23).

∗∆∗+−∆+°∆∗−∆=°∆

opToTo T

TTToTCSTHG ln)( pC

where T is the temperature in Kelvin (K)

To is an arbitrary reference temperature (e.g. 298.15 K)

∆HTo is the enthalpy change upon binding at To (kcal.mol-1)

∆SºTo is the standard state entropy change upon binding at To (kcal.mol-1)

and ∆Cp is the specific heat capacity (kcal.mol-1.K-1), and is assumed to be temperature-

independent.

- 36 -

The ∆Cp is a measure of the dependence of ∆H (and ∆S) on temperature. It is almost invariably

negative for protein/protein interactions (24), indicating that enthalpic effects become more

favourable and entropic effects less favourable as temperature increases. This negative heat capacity

is believed to be the result of the disruption at high temperatures of the ordered 'shell' of water that

forms over the non-polar surfaces of a macromolecule. Consequently, the favourable entropic effect

of displacing the shell upon binding is reduced. And because fewer solvent bonds are disrupted at the

higher temperature the net enthalpy change becomes more favourable. ∆Cp is a useful measure of the

extent of non-polar surface that is buried upon binding (25). However determining ∆Cp by van't Hoff

analysis is likely to be inaccurate. A second drawback of van't Hoff analysis is that changes in

temperature may also affect the interactions between the proteins and the solution components,

including water (26). If these equilibria are coupled to the protein/protein interaction

they will contribute to the ∆HvH, which will therefore differ from the ∆H determined by calorimetry.

Because of these drawbacks, it is advisable to confirm ∆H and ∆Cp determinations by calorimetry.

Unfortunately even recently developed microcalorimeters require about one hundred fold more

protein than the BIAcore. Thus the BIAcore may be the only means of obtaining enthalpy and heat

capacity data when limited amounts of material are available.

7.5. Activation energy

The kon and koff will generally increase with temperature. The extent of this increase is a measure of

the amount of thermal energy required for binding or dissociation, and is referred to as the activation

energy of association (Eaass) or dissociation (Ea

diss). Ea can be determined using the Arrhenius

equation. Assuming that Ea is constant over the temperature range examined, then

TREAk a

∗−= lnln

where k is the relevant rate constant (e.g. kon and koff), R is the gas constant, and A is a constant

known as the pre-exponential factor. Ea is determined from the slope of a plot of lnk versus 1/T.

Importantly, because Eaass and Ea

diss can be considered activation enthalpies, the reaction enthalpy can

be calculated from the relationship

∆H = Eaass - Ea

diss

An unusually high Ea value indicates that binding and/or dissociation require the surmounting of high

- 37 -

potential energy barriers, suggesting that conformational rearrangements are required.

8. Conclusion

SPR provides a powerful tool for the analysis of protein/protein interactions. This is particularly true

for low affinity interactions, which are difficult to study using any other technique. One of the most

useful features of SPR is that it provides, through binding analysis, a quick way of checking the

structural integrity of recombinant molecules. SPR is also useful for measuring the affinity, enthalpy,

stoichiometry, kinetics and activation energy of an interaction. A major advantage of SPR over other

techniques such as calorimetry is that much smaller amounts of protein are required. The pitfalls

associated with SPR are easily avoided once they are understood. SPR is not well-suited to high-

throughput assays, or the analysis of small molecules (Mr < 1000).

9. Appendix

9.1. Physical basis of SPR

When a beam of light passes from material with a high refractive index (e.g. glass) into material with

a low refractive index (e.g. water) some light is reflected from the interface. When the angle at which

the light strikes the interface (the angle of incidence or θ) is greater than the critical angle (θc), the

light is completely reflected (total internal reflection). If the surface of the glass is coated with a thin

film of a noble metal (e.g. gold), this reflection is not total; some of the light is 'lost' into the metallic

film. There then exists a second angle greater than the critical angle at which this loss is greatest and

at which the intensity of reflected light reaches a minimum or 'dip'. This angle is called the surface

plasmon resonance angle (θspr). It is a consequence of the oscillation of mobile electrons (or 'plasma')

at the surface of the metal film. These oscillating plasma waves are called surface plasmons. When

the wave vector of the incident light matches the wavelength of the surface plasmons, the electrons

- 38 -

'resonate', hence the term surface plasmon resonance. The 'coupling' of the incident light to the

surface plasmons results in a loss of energy and therefore a reduction in the intensity of the reflected

light. It is because the amplitude of the wave vector in the plane of the metallic film depends on the

angle at which it strikes the interface that an θspr is observed. An evanescent (decaying) electrical

field associated with the plasma wave travels for a short distance (~300 nm) into the medium from

the metallic film. Because of this, the resonant frequency of the surface plasma wave (and thus θspr)

depends on the refractive index of this medium. If the surface is immersed in an aqueous buffer

(refractive index or µ ~1.0) and protein (µ ~1.33) binds to the surface, this results in an increase in

refractive index which is detected by a shift in the θspr. The instrument uses a photo-detector array to

measure very small changes in θspr. The readout from this array can be viewed on the BIAcore as

'dips' (Section 3.3). The change is quantified in resonance units or response units (RUs) with 1 RU

equivalent to a shift of 10-4 degrees. Empirical measurements have shown that the binding of 1

ng/mm2 of protein to the sensor surface leads to a response of ~1000 RU. Since the matrix is ~100

nm thick, this represents a protein concentration within the matrix of 10 mg/mL. Apart from the

refractive index, the other physical parameter which affects θspr is temperature. Thus a crucial feature

of any SPR instrument is precise temperature control.

9.2. Samples and buffers

9.2.1. Guidelines for preparing samples and buffers for use on the BIAcore

• All buffers should be filtered through 0.2 µm filters and degassed at room temperature. The latter

can be achieved by filtering under vacuum or by using a vacuum chamber.

• All samples >3 mL should be filtered and degassed in the same way.

• If samples <3 mL should be spun at high speed in a microcentrifuge for 10-20 min at 4 C and

- 39 -

then degassed in a vacuum chamber.

• Before samples are placed in the sample rack they should be pulsed briefly in a microcentrifuge.

This dislodges air-bubbles from the bottom of the container, helps ensure that the meniscus is

horizontal.

• Vials should be capped to prevent sample evaporation.

• When running long experiments consider cooling the sample rack base using a thermostatic re-

circulator.

9.2.2. Standard buffers

9.2.2.1. Running buffers

HBS or HBS-EP: 10 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% P20

HBS-P: 10 mM Hepes pH 7.4, 150 mM NaCl, 0.005% P20

HBS-N: 10 mM Hepes pH 7.4, 150 mM NaCl

9.2.2.2. Pre-concentration buffers

pH 3.0-4.5 10 mM formate

pH 4.0-5.5 10 mM acetate

pH 5.5-6.0 5 mM maleate

9.2.2.3. Regeneration buffers

Grouped according to chemical properties. They increase in strength from left to right.

Cation chelator: HBS with 20 mM EDTA pH 7.5

High ionic strength: NaCl 1 M; KCl 4 M; MgCl2 2 M

Low pH: Glycine/HCl 100 mM pH 2.5; HCl 10 mM; HCl 100 mM; H3PO4 100 mM

High pH: NaOH 5 mM; NaOH 50 mM

- 40 -

9.3. Additional information

BIAcore maintains a useful web site [http://www.biacore.com/]. This announces new product

developments and includes a continuously updated list of SPR publications and an electronic version

of the BIAjournal.

The handbooks that come with the instrument are an essential resource. These can also be purchased

from BIAcore along with the following books, which describe the technology and its applications in

more detail.

BIAtechnology Handbook

BIAapplication Handbook

BIAevaluation software Handbook

10. References

1. Jönsson, U., Fägerstam, L., Ivarsson, B., Johnsson, B., Karlsson, R., Lundh, K., Löfås, S.,

Persson, B., Roos, H., Rönnberg, I., Sjölander, S., Stenberg, E., Ståhlberg, R., Urbaniczky, C.,

Östlin, H., and Malmqvist, M. (1991) BioTechniques 11(5), 620

2. Jönsson, U., and Malmqvist, M. (1992) Advances in Biosensors 2, 291

3. Hodgson, J. (1994) Bio/Technology 12, 31

4. van der Merwe, P. A., and Barclay, A. N. (1996) Current Opinion in Immunology 8, 257

5. Schuck, P. (1997) Annu Rev Biophys Biomol Struct 26, 541

6. O'Shannessy, D. J. (1994) Current Opinion in Biotechnology 5, 65

7. van der Merwe, P. A., Brown, M. H., Davis, S. J., and Barclay, A. N. (1993) Biochem. Soc.

Trans 21, 340S

8. Myszka, D. G. (1997) Curr Opin Biotechnol 8(1), 50

9. Karlsson, R., and Falt, A. (1997) J Immunol Methods 200(1-2), 121

10. van der Merwe, P. A., and Barclay, A. N. (1994) Trends Biochem. Sci. 19, 354

11. Wyer, J. R., Willcox, B. E., Gao, G. F., Gerth, U. C., Davis, S. J., Bell, J. I., van der Merwe, P.

A., and Jakobsen, B. K. (1999) Immunity 10, 219

- 41 -

12. Willcox, B. E., Gao, G. F., Wyer, J. R., Ladbury, J. E., Bell, J. I., Jakobsen, B. K., and van der

Merwe, P. A. (1999) Immunity 10, 357

13. van der Merwe, P. A., Crocker, P., Vincent, M., Barclay, A. N., and Kelm, S. (1996) Journal of

Biological Chemistry 271, 9273

14. van der Merwe, P. A., McNamee, P. N., Davies, E. A., Barclay, A. N., and Davis, S. J. (1995)

Current Biology 5, 74

15. Davis, S. J., Davies, E. A., Tucknott, M. G., Jones, E. Y., and van der Merwe, P. A. (1998)

Proc. Natl. Acad. Sci. USA 95, 5490

16. van der Merwe, P. A., Bodian, D. L., Daenke, S., Linsley, P., and Davis, S. J. (1997) J. Exp.

Med. 185, 393

17. van der Merwe, P. A., Brown, M. H., Davis, S. J., and Barclay, A. N. (1993) EMBO J 12, 4945

18. van der Merwe, P. A., Barclay, A. N., Mason, D. W., Davies, E. A., Morgan, B. P., Tone, M.,

Krishnam, A. K. C., Ianelli, C., and Davis, S. J. (1994) Biochemistry 33, 10149

19. Karlsson, R., and Ståhlberg, R. (1995) Anal. Biochem. 228, 274

20. Nicholson, M. W., Barclay, A. N., Singer, M. S., Rosen, S. D., and van der Merwe, P. A.

(1998) J. Biol. Chem. 273, 763

21. Felder, S., Zhou, M., Hu, P., Ureña, J., Ullrich, A., Chaudhuri, M., White, M., Shoelson, S. E.,

and Schlessinger, J. (1993) Mol Cell Biol 13(3), 1449

22. Myszka, D. G., and Morton, T. A. (1998) Trends in Biochemical Sciences 23, 149

23. Yoo, S. H., and Lewis, M. S. (1995) Biochemistry 34, 632

24. Stites, W. E. (1997) Chem. Rev. 97, 1233-1250

25. Ladbury, J. E., and Chowdhry, B. Z. (1996) Chem Biol 3(10), 791

26. Naghibi, H., Tamura, A., and Sturtevant, J. M. (1995) Proc Natl Acad Sci U S A 92(12), 5597

11. Acknowledgements

I would like to thank Liz Davies, Neil Barclay, and Don Mason for critical comments on the

manuscript. This work was supported by the Medical Research Council.

12. GLOSSARY

- 42 -

KD dissociation constant (M)

kon association rate constant (M-1.s-1)

koff dissociation rate constant (s-1)

mAb monoclonal antibody

RU response units or resonance units

SPR surface plasmon resonance

- 43 -

13. Figure 1. Analysis of dips.

Several examples are shown of normal (1 and 2) and abnormal (3 and 4) dips. These are discussed in

Protocol 1 .

- 44 -

14. Tables

14.1. Table 1. BIAcore instruments currently available (January 1999)

BIAcore X BIAcore2000 BIAcore3000

Automated No Yes Yes

Temperature control (°C) 10 below ambient to

40

4 to 40 4 to 40

Flow-cell number 2 4 4

Flow-cell volume (nL) 60 60 20

Time resolution (Hz)

single flow cell

two flow cells

10

5

10

2.5

10

5

Refractive index range 1.33-1.40 1.33-1.36 1.33-1.40

Sample recovery Manual Automatic

Fraction collection Manual Automatic

Collects surface bound

material only, but in very

small volume (3-7 µL)

Baseline noise

(RU, Root Mean Square )

0.3 0.3 0.1

Online subtraction of

background response

Yes Yes (flow cells 2-1,

3-1, 4-1)

Yes (flow cells 2-1, 3-1, 4-

1, 3-4)

- 45 -

14.2. Table 2. Sensor chips available for use on the BIAcore

Surface chemistry Free

carboxyme

thyl

groups

Comments

Sensor Chip CM5

(research grade)

carboxymethylated

dextran matrix

Yes Most widely used sensor chip. Suitable for most

applications.

Sensor Chip CM5

(certified grade)

as above Yes Less chip-to-chip variation than research grade

chip but much more expensive.

Sensor chip SA as above with

streptavidin pre-

coupled

No Quite expensive, especially since it cannot be re-

used. It is cheaper to couple streptavidin to CM5

research grade chip (11).

Sensor chip NTA As above with NTA

pre-coupled

No Comes with nitrilotriacetic acid (NTA) coupled.

For capturing proteins with oligo-histidine tags.

Expensive but can be re-used many times.

Sensor chip HPA Flat hydrophobic

surface

No For immobilising lipid monolayers. Problem

with non-specific binding.

Pioneer Chip C1 No dextran matrix.

Carboxymethylated

Yes For binding particles (eg cells) too large to enter

dextran matrix. Also helps eliminate potential

matrix-related artefacts.

Pioneer Chip B1 Low level of

carboxymethylation

Yes Its lower charge density makes it useful

reducing non-specific interactions with charged

analytes.

Pioneer Chip F1 Thin dextran matrix Yes For binding to bulky particles. Reduces matrix-

related artefacts.

Pioneer Chip J1 Unmodified gold

surface

No Can be used to design novel coupling chemistry

Pioneer Chip L1 Dextran derivatized

with lipophillic

compounds

No For immobilizing liposomes via interactions

with lipophilic compounds. Apparently lower

levels of non-specific binding than HPA chip.

- 46 -

14.3. Table 3. Covalent coupling chemistry

Surface activationa Group required on protein Protein preparation Comments

Amine EDC/NHSb

Amine groups (lysine and

unblocked N-termini)

No preparation needed. Can be used with most

proteins. Risk of multiple

coupling.

Surface-

thiol

EDC/NHS followed by

cystamine/DTT to

introduce a thiol group

Amine or carboxyl groups. Need to introduce reactive

disulphide using heterobifunctional

reagent such as PDEA (carboxyl) or

SPDP (amine).

Not widely used

Ligand-thiol EDC/NHS followed by

PDEA to introduce a

reactive disulphide

group

Surface exposed free cysteine or

disulphide.

Reduce disulphides under non-

denaturing conditions to generate

free cysteine.

Useful for proteins with

exposed disulphides or free

cysteines. Multiple

coupling unlikely.

Aldehyde

coupling

EDC/NHS followed by

hydrazine

Aldehyde groups. Create aldehydes by oxidising cis-

diols (in sugars) with periodate.

Works especially well with sialic

acid.

Useful for polysaccharides

and glycoproteins.

a All coupling reactions utilise carboxymethyl groups on the sensor surface and can be used with any sensor chip which has free

carboxymethyl groups (Table 2). The first step is to activate these groups with N-hydroxysuccinimide, thus creating a highly reactive

succinimide ester which reacts with amine groups on protein, or can be further modified. b Abbreviations: EDC, N-ethyl-N'-(dimethylaminopropyl)carbodiimide; NHS, N-hydroxysuccinimide; DTT, dithiothreitol; PDEA, 2-(2-

pyridinyldithio)ethane-amine.

- 47 -

14.4. Table 4. Techniques for ligand capture.

Covalently

coupled molecule Captured ligand Valency of ligand Removal of

Tag Comments

Antibodies

Rabbit-anti mouse Fc polyclonal (26)

Any mouse IgG monoclonal antibody

divalent no Antibody available from BIAcore. Because it is polyclonal very high levels of binding to a particular isotype are difficult to achieve

Mouse anti-human Fc, monoclonal (R10Z8E9) (13,27)

Any protein fused to the Fc portion of human IgG1. This includes most Fc chimeras.

divalent notroutinely. However new vectors incorporate

Antibody available from Recognition Systems, University of Birmingham Science Park, Birmingham B15 2SQ, U.K.

Mouse anti rat CD4, monoclonal (OX68) (17)

Any protein fused to rat CD4 domains 3 and 4.

monovalent no Antibody available from Serotec.

Anti-GSTb Any protein expressed as a GST chimera

divalent yes Antibody available from BIAcore. Because GST dimerizes, the fusion proteins are dimers.

Other Streptavidin Any biotinylated molecule. Biotinylation can be indiscriminate or (preferably) targeteda.

monovalent, can be made tetravalent with streptavidin

no Steptavidin sensor chips are available from BIAcore. However streptavidin can be coupled to standard CM5 chips at lower cost (11). Note that streptavidin surfaces cannot be regenerated.

Ni-NTA Any protein with oligo histidine tag

monovalent yes Sensor chips can be purchased from BIAcore with Ni-NTA already coupled.

aTargeted biotinylation involves introducing a single biotin group on the molecule in a position in which it will not interfere with binding to analyte. This can be achieved either by chemical synthesis (e.g. oligonucleotides), or by enzymatic biotinylation. An established method uses the enzyme BirA to biotinylated a specific peptide which has been added on to the N or C terminus of a recombinant protein (11). bAbbreviations: GST, glutathione-S-transferase; Ni-NTA, nickel-nitrilotriacetic acid

- 48 -

14.5. Table 5. Trivial causes of complex binding kinetics

Cause How to detect and/or eliminate

Mass-transport limitations • Increase the flow-rate and lower the density of immobilised

ligand

• Alternatively, include mass-transport term in fitting equation.

Drifting baseline • Should be evident in the control sensorgrams.

• Try to eliminate this by subtracting the control sensorgram.

• Alternatively, the simple 1:1 binding model can be modified to

include a drifting baseline.

Bulk refractive index artefacts • Should be evident in the control sensorgrams.

• Try to eliminate this by subtracting the control sensorgram.

• Alternatively, the simple 1:1 binding model can be modified to

include bulk refractive index artefacts.

Rebinding • The fit gwill be worse at higher levels of immobilization.

• Increase the flow-rate and lower the density of immobilised

ligand.

• Inject a competing small analyte during the dissociation phase

(21).

Heterogeneous immobilisation • Immobilise ligand in a different way (preferably indirectly, by

ligand capture).

Analyte is multimeric • Perform size-exclusion chromatography and/or analytical

ultracentrifugation to ensure that analyte is monomeric.

• If the ligand is monomeric, perform kinetic analysis in reverse

orientation.

Analyte is monomeric but

contaminated by multivalent

aggregates

• Repeat the experiment using the monomeric fraction

immediately after size-exclusion chromatography, avoiding

concentrating or freezing the sample.

- 49 -

14.6. Table 6. Distinguishing some non-trivial causes of complex kinetics

Binding

mechanism

Description Distinguishing features

Heterogenous

analyte

More than one form of the

analyte binds to a single ligand.

Dissociation will slow as the duration of

the injection is increased. This effect will

not be seen in the reverse orientation.

Heterogenous

ligand

One analyte binds to more than

one ligand

Dissociation will not be affected by the

duration of the injection. However, in the

reverse orientation dissociation will slow as

the duration of the injection is increased.

Two-state

binding

(conformational

change)

After forming, the

ligand/analyte complex

interconverts between two or

more forms with different

kinetic properties.

The dissociation phase will slow as the

duration of the injection is increased. This

affect will also be seen in the reverse

orientation.

- 50 -